The objectives of this proposal are to develop an exciting new assay format for detecting and quantifying the action of single biomolecules. The specific aims are to develop a prototype assay, and to determine the limits of sensitivity for detecting and quantifying the presence of proteases and transcription factor ligands. The innovative technology is based on the use of a novel reporter label which allows the monitoring and quantifying the action of enzymes on individual molecule substrates. This new approach is ideally suited for miniaturization, and will be developed for both ultra-high throughput screening (UHTS) for new drugs, and for medical diagnostic assays. We expect the results of our SBIR program, in collaboration with industrial partners, to allow us to create commercial products for sale to both the research and clinical biomedical communities. Additional products will be developed for the detection of low levels of toxic agents (BCD), monitoring environmental contaminants, and measurement of illegal drug levels. PROPOSED COMMERCIAL APPLICATION: The manufacture and sale of proprietary reagents, kits, and associated consumables needed to perform the assays described. In addition, a fully automated system comprising a microscope, light source, detection device, and image analysis software suitable for high throughput screening applications.